Short Protein Evolution Stories: Illuminating Aspects of MempromCC and Histidine Kinase Evolution

Abstract

Sequence-based bioinformatic analysis has long been a driving force of evolutionary studies, while experimental approaches offer new insights through mutagenesis, in vivo and in vitro evolution. Here, we employ both approaches to illuminate aspects of the evolution of two different protein families, the mempromCC protein family and the histidine kinase protein family. The mempromCC family comprises membrane-bound coiled-coil-containing proteins found mostly in mitochondria. Our bioinformatic analysis connects for the first time these proteins allowing their collective structural and functional analysis. While the mechanism of function remains elusive, these proteins have been shown to act as assembling factors of different proteins like the mitochondrial Ca2+ uniporter (MCU), cytochrome c and Photosystem I that are crucial for cell survival. Histidine kinase proteins typically function as multidomain proteins, comprising transmembrane sensor and cytoplasmic effector domains. In the osmoregulating histidine kinase EnvZ, signals are transmitted through the DHp (dimerization and histidine phosphotransfer) and the CA (catalytic and ATPbinding) domains: CA carries ATP and phosphorylates a histidine in DHp, which then transfers the phosphate group to downstream effectors, resulting in a modulated transcription of genes controlled by the ompC promoter. Based on the evolutionary traits observed for both DHp and CA an evolutionary scenario has emerged, in which the two domains were fused early in evolution building a histidine kinase from a simple ATP-binding element. To study this possibility, we produced a chimera, in which CA was replaced with DX, an artificial protein that was generated through in-vivo evolution by selecting for ATP binding affinity. Indeed, a DHp-DX fusion protein (DHp-DX1) showed a strong ATPase activity in vitro, that was not seen for DX alone. In vivo, DHp-DX1 increased the transcription of ompC-controlled genes compared to a phosphotransferasedeficient DHp-DX1(H15Q) mutant. Collectively, these findings support the modular evolution of the DHp-CA element and offer a proof of concept for primordial enzyme evolution.